The goal of the proposed research is to determine mechanisms of nonenveloped virus internalization, disassembly, and membrane penetration using mammalian reovirus as an experimental system. Given its capacity to target multiple tissue types and organs in mice, reovirus provides an attractive experimental platform to determine the role of viral entry steps in disease pathogenesis. Reovirus entry requires ?1 integrin-mediated internalization, proteolytic removal of outer-capsid protein ?3, and penetration of endosomal membranes by outer-capsid protein ?1. As with many other pathogenic viruses, the biochemical processes that regulate these progressive events in reovirus entry are not understood, nor is it known how the entry steps influence the course of reovirus disease. These gaps in knowledge have hindered efforts to develop antivirals that act at the earliest stages of infection and highly selective viral vectors for gene-delivery applications. Three specific aims are proposed to elucidate mechanisms of reovirus entry into cells. In Specific Aim 1, the means by which ?1 integrin promotes reovirus internalization and endocytic transport will be determined. Mutant viruses with alterations in integrin-binding motifs (IBMs) will be engineered using a newly developed plasmid-based reverse genetics system and tested for the capacity to bind recombinant ?1 integrin and infect cells. Mechanisms of ?1 integrin-mediated virus uptake and endocytic transport will be defined using pathway-specific inhibitors, infectivity assays, and cell imaging. The role of ?1 integrin in reovirus pathogenesis will be determined using mice expressing mutant ?1 integrin and IBM mutant viruses. In Specific Aim 2, viral determinants of reovirus disassembly and membrane penetration will be identified. Mutant viruses with structure- guided alterations in ?3 and ?1 will be generated by reverse genetics and assessed for disassembly, membrane penetration, and infection. The role of endocytic proteases cathepsins B, L, and S in reovirus pathogenesis will be defined using cathepsin-deficient mice and cathepsin inhibitors. In Specific Aim 3, the structural basis for reovirus disassembly and membrane penetration will be elucidated. Structures of sequential reovirus disassembly intermediates will be determined at subnanometer resolution using cryo-EM and 3D image reconstruction. Structures of a protease-hypersensitive ?3 mutant and the ?1 ? domain, which is required for membrane penetration, will be determined by X-ray crystallography. This research will reveal how viral and cellular proteins cooperate in a highly ordered biochemical pathway that culminates in cell entry by a nonenveloped virus. These studies also will illuminate new targets for therapy against pathogens that use the endocytic pathway and cathepsin proteases to parasitize host cells.